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Impedance Spectroscopy for Silica Nanoparticle Detection in Caco-2 Cells
Available online at www.sciencedirect.com
ScienceDirect Procedia Engineering 87 (2014) 364 – 368
EUROSENSORS 2014, the XXVIII edition of the conference series
Impedance spectroscopy for silica nanoparticle detection in Caco-2 cells S. Claraa, M. R. Lornejad-Schäferb*, C. Schäferb, B. Jakobya and W. Hilbera a
Institute for Microelectronics and Microsensors, Johannes Kepler University, Linz, Austria b BioMed-zet Life Science GmbH, Industriezeile 36/VII, A-4020 Linz, Austria
Abstract We present a feasibility study aiming at the detection of silica nanoparticles (NPs) in human intestinal epithelial cells. Caco-2 cells were maintained in cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM) and differentiated for 21 days. To study the effect of silica NPs on the differentiated Caco-2 cell, cells were treated for 24h with different doses. We measured the serum-free culture medium first, which was supplemented with different doses of silica NPs using a parallel-plate capacitor principle to demonstrate the sensitivity of the sensing principle to the particle concentration. In a next step we treated Caco-2 cells with serum-free culture medium containing silica NPs. After 24h incubation time, culture medium was removed and cells were washed with PBS, so that topical adherent NPs should have been removed. We finally present the use of a second coplanar electrode design with eight single electrodes connectable in arbitrary combination to detect the silica NPs in cells. © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2014 The Authors. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the scientific committee of Eurosensors 2014. Peer-review under responsibility of the scientific committee of Eurosensors 2014 Keywords: impedance spectroscopy, silica nanoparticles, caco-2 cells
1. Introduction Silica nanoparticles (NPs) are common in our everyday life e.g. they can be found in cosmetics, printer toners, glass products, and our food and their intake from known and unknown sources could be harmful ([1], [2]). In foodstuff, for instance, they can occur as flow aid in powder sugar, salt and coffee creamer and daily intake into the
* Corresponding author. Tel.: +43732770325; fax: +4373277032513. E-mail address: [email protected]
1877-7058 © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the scientific committee of Eurosensors 2014 doi:10.1016/j.proeng.2014.11.741
365
S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
human body so far, can only be estimated. After ingestion of Silica NPs from known and unknown sources they occur in the gastro-intestinal tract and affect or damage the intestinal cells. As presented in [3] in many epithelial cells NPs are taken up and are able to cross the cell membrane and become internalized. After uptake they can accumulate in certain cell regions. However, accurate detection and quantification of Silica NPs that are located inside the cells using flexible and cheap measurement methods is lacking but urgently needed for risk assessment and maintenance of human’s health. A new appropriate impedance spectroscopy-based device could be applicable for this purpose.
a)
b)
Fig. 1. (a) Parallel-plate capacitor design used for reference measurements of the cell-free medium; (b) Spring probe, the holder with the spring probes can be connected directly to the 24 well plate and contacts every electrode individually. The switching electronics, which is not shown in this image, allows connecting the electrodes in any combination to the impedance measurement system.
2. Measurement procedure In this feasibility study, we first show the sensitivity of the method to detect silica NPs that are dispensed into the culture medium in different concentrations. For this purpose we used a simple two plate capacitor configuration (Fig. 1a) immersed directly into the beaker containing the suspension. For the experimental proof of NPs ingestion into cells we fabricated special coplanar electrodes (see Fig. 2) described below and fixed them on the bottom of each well of the 24-well plates. After that the wells were seeded with caco-2 cells maintained in a cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM). After 21 days of differentiation the cells are accrued on the electrodes (see Fig. 2c). Then the cells were treated with different Silica NPs concentrations from 25 μg/ml to 500 μg/ml for 24h. After that, the culture media was removed, and the cells were washed using Phosphate Buffered Saline (PBS) buffer to remove possible sedimented NPs from the cell surface. Next the coplanar electrodes were contacted with the spring probe (see Fig. 1b) and the electrical impedance was measured for various electrode combinations (see Fig. 3c).
II I a)
b)
c)
Fig. 2. (a) Shows the electrode design and the naming of the different electrodes; (b) Shows a detailed view of the electrodes containing the dimensioning; (c) Shows the Caco-2 cells grown on the electrodes. I) with Au and II) without Au
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S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
3. Electrode design For the measurements a special electrode design was used to simplify the handling and offer the possibility to measure various positions in one sample without changing the electrode or the connections. The electrode itself is shown in Fig. 1 and consists of eight individually connectable electrodes which allow various electrode combinations for measurements at different sample areas. For the fabrication of the electrodes a 0.1 mm thick Polyethylenterephthalat (PET) film was used as a substrate. Circular shaped elements which directly fit into the 24well plate were cut out of the PET foil and a shadow mask was used to directly evaporate 30 nm thick gold (Au) structures as the measurement electrodes on them. The biocompatibility of the Au-electrodes was approved by the results from cytotoxicity biochemical assay (Lactate dehydrogenase (LDH) release). The segmented contact pads for the connections of the measurement electronics are placed on the outer boundary of the circular substrates and can be contacted with spring probes. In the middle of the electrode substrate is a transparent region to allow the use of a transmitted light microscope, which is very important for an optical control during the growth phase (see Fig. 2c). The electrodes are placed directly on the bottom of the 24-well plate. The electrical connection of the substrate is performed using eight spring probes arranged in a circle (see Fig. 1b) and mounted into a buckler. The buckler fits on the used 24-well plate and guaranties a good connection of the spring probes on the gold electrodes. 4. Readout electronics For the measurement of the impedance spectrum an Agilent 4294A Precision Impedance Analyzer was used. This measurement system works with an auto balancing bridge principle [4] and provides a four terminal connection interface. This interface has two connectors for the current path (HC and LC) and two connectors for the potential measurements (HP and LP). Between the HC and the LC connectors an alternating current with constant amplitude is applied by the measurement system. The other two ports (HP and LP) are used for the potential measurements. To keep the advantages of the four terminal measurement principle the connectors H C and HP should be connected together as close to the device under test (DUT) as possible, the same applies also for the LC and LP connectors. For the implementation of the automatic connection of arbitrary electrode combinations, two matrix switches from Maxim Integrated were used (MAX14661). This 16:2 analog matrix switch allows it to connect each of the 16 pins to any of the common pins simultaneously in any combination [5]. For the circuit all of the four terminal connections need to be switchable in any combination, therefore two of the matrix switches were combined to a 16:4 system. This connection method allows neglecting the “on” resistance RON and keeping the advantages of a four wire measurement over a large frequency range (40 Hz up to 10 MHz). For the measurement it is important to keep the current over the matrix switch below the maximum rating of 50 mA to guarantee correct measurement results. The control of the measurement system was implemented in MATLAB controlling the impedance analyzer via an Ethernet interface to allow an automatic sequence of impedance measurements for various electrode combinations. The communication with the matrix switches was implemented using a μController as USB to Serial Peripheral Interface (SPI) converter. 5. Measurements As a first measurement the direct influence of the nanoparticles in the medium was measured with a parallel-plate capacitor and compared to the results of the coplanar electrode setup. Fig 3 a) and b) shows the results for different concentrations of NPs with both setups. As mentioned above, the coplanar setup allows for a large variety of electrode connections. The electrodes a, b, c and d are “center electrodes”, i.e. they form a segmented ring in the center of the well (see Fig. 2a). The other electrodes e, f, g and h form are placed outside of these center electrodes as shown in Fig. 2a. Table 1 summarizes the used options in measuring impedances between two electrodes or groups of these electrodes. In case of “single electrode combinations” two single electrodes are selected and the impedance between them is measured where only the center electrodes are used. In case of “double electrode combinations” the impedance between a single electrode and two other connected neighboring electrodes is
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S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
determined. In that manner, the measurement area in the well can be spatially shifted. In the measurements given in Fig 3b the average of single electrode measurements in group A (see Table 1) is shown which represents the situation at the center of the well. In Fig 3c, as an example for the spatial variation, all impedances are shown in one plot. The spectra for each group (A, B, C, D and E, see Table 1) are shown in the same color and they virtually coincide such that they appear as a single characteristic. Table 1. Measured electrode combinations. Combinations
Groups A)
Single electrode combinations B)
C)
gap between adjacent center electrodes gap between opposite center electrodes
Two center electrodes
Double electrode combination D)
Full electrode measurement
a)
E)
One center electrode
All electrodes
b)
Number in sequence
Terminals: HC, HP
Terminals: LC, LP
1
a
b
2
b
c
3
c
d
4
d
a
5
a
c
6
b
d
7
a, b
e
8
b, c
f
9
c, d
g
10
d, a
h
11
a
h, e
12
b
e, f
13
c
f, g
14
d
g, h
15
a, b, c, d
e, f, g, h
c)
Fig. 3. (a) Measurement results using the parallel-plate capacitor sensor setup shown in Fig. 2 a) for the nanoparticles dispensed into the culture medium with different concentrations at 3.2 kHz; (b) Measured impedance spectra for different nano particle concentrations using the coplanar electrode configuration group A (see Table 1); (c) Measurement for all different electrode configurations specified in Table 1 for one sample (particle concentration 50 μg/ml).
It is clearly visible that the parallel-plate capacitor provides more sensitive results to the particle concentration whereas the sensitive area of the coplanar design is concentrated in the gap between the electrodes thus yielding smaller signals. However, with the co-planar electrode arrangement not the whole volume is sensed but only a small layer above the electrodes which allows to detect a potentially non-uniform spatial distribution of the cells, which was not present in the measurement shown, though. This present uniformity in the particle and cell distribution is also indicated by the aforementioned coinciding spectra within each group (A, B, C, D, and E) of electrode combinations as shown in Fig 3c.
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S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
A disadvantage of the measurements method is that, in the current design, the connections to the coplanar electrodes by means of the spring probes are realized in the sample, i.e. at the bottom of the well. Therefore the reproducibility cannot be guaranteed and the measurements for different equally filled wells tend to vary by random offsets due to variations in contact resistances. 6. Conclusion We presented measurement results for different doses of silica NPs supplemented in the cell culture media, measured with both the parallel-plate capacitor and the coplanar electrode design. Both type of electrodes show a clear dependency of the frequency dependent electrical impedance on the NPs concentration where the parallel-plate capacitor design provides more reproducible results due to the more homogeneous field distribution and the averaging over a relatively large volume. The measurements with the coplanar electrodes offer the possibility of spatially resolved measurements in small sample wells and hence to probe spatially defined areas of the cell culture, but suffer from the fact that the electrical connections between spring probes and electrodes can be affected by sample residues in the contact area. 7. Outlook The previously mentioned problems with the connection of the electrodes and the hardly controllable deviations in contact resistance and in cell population between the substrates in different wells require the development of an improved measurement principle where each of the cell culture wells is monitored separately over the time. This measurement method will allow using the relative changes of the spectra to monitor the cell behavior in response to NPs. Acknowledgements This work was supported by the “REGIO 13 - Impulse für OÖ” program, the “Austrian Center of Competence in Mechatronics” (ACCM) funded by the Austrian Research Promotion Agency (FFG) and by State of Upper Austria. References [1] [2] [3]
[4] [5]
EFSA - European Food Safety Authority. 2009. The potential risks arising from nanoscience and nanotechnologies on food and feed safety (EFSA-Q-2007-124a). The EFSA Journal 958, 1-39 Moghimi SM, Hunter AC, Murray JC: Nanomedicine: current status and future prospects. FASEB J 2005, 19:311-330. Environ Sci Technol. 2005 Dec 1;39(23):9370-6. Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Limbach LK1, Li Y, Grass RN, Brunner TJ, Hintermann MA, Muller M, Gunther D, Stark WJ Agilent Impedance Measurement Handbook A guide to measurement technology and techniques 4th Edition, 2009-2013 Datasheet: MAX14661 Beyond-the-Rails 16:2 Multiplexer, maxim integrated
ScienceDirect Procedia Engineering 87 (2014) 364 – 368
EUROSENSORS 2014, the XXVIII edition of the conference series
Impedance spectroscopy for silica nanoparticle detection in Caco-2 cells S. Claraa, M. R. Lornejad-Schäferb*, C. Schäferb, B. Jakobya and W. Hilbera a
Institute for Microelectronics and Microsensors, Johannes Kepler University, Linz, Austria b BioMed-zet Life Science GmbH, Industriezeile 36/VII, A-4020 Linz, Austria
Abstract We present a feasibility study aiming at the detection of silica nanoparticles (NPs) in human intestinal epithelial cells. Caco-2 cells were maintained in cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM) and differentiated for 21 days. To study the effect of silica NPs on the differentiated Caco-2 cell, cells were treated for 24h with different doses. We measured the serum-free culture medium first, which was supplemented with different doses of silica NPs using a parallel-plate capacitor principle to demonstrate the sensitivity of the sensing principle to the particle concentration. In a next step we treated Caco-2 cells with serum-free culture medium containing silica NPs. After 24h incubation time, culture medium was removed and cells were washed with PBS, so that topical adherent NPs should have been removed. We finally present the use of a second coplanar electrode design with eight single electrodes connectable in arbitrary combination to detect the silica NPs in cells. © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2014 The Authors. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the scientific committee of Eurosensors 2014. Peer-review under responsibility of the scientific committee of Eurosensors 2014 Keywords: impedance spectroscopy, silica nanoparticles, caco-2 cells
1. Introduction Silica nanoparticles (NPs) are common in our everyday life e.g. they can be found in cosmetics, printer toners, glass products, and our food and their intake from known and unknown sources could be harmful ([1], [2]). In foodstuff, for instance, they can occur as flow aid in powder sugar, salt and coffee creamer and daily intake into the
* Corresponding author. Tel.: +43732770325; fax: +4373277032513. E-mail address: [email protected]
1877-7058 © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the scientific committee of Eurosensors 2014 doi:10.1016/j.proeng.2014.11.741
365
S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
human body so far, can only be estimated. After ingestion of Silica NPs from known and unknown sources they occur in the gastro-intestinal tract and affect or damage the intestinal cells. As presented in [3] in many epithelial cells NPs are taken up and are able to cross the cell membrane and become internalized. After uptake they can accumulate in certain cell regions. However, accurate detection and quantification of Silica NPs that are located inside the cells using flexible and cheap measurement methods is lacking but urgently needed for risk assessment and maintenance of human’s health. A new appropriate impedance spectroscopy-based device could be applicable for this purpose.
a)
b)
Fig. 1. (a) Parallel-plate capacitor design used for reference measurements of the cell-free medium; (b) Spring probe, the holder with the spring probes can be connected directly to the 24 well plate and contacts every electrode individually. The switching electronics, which is not shown in this image, allows connecting the electrodes in any combination to the impedance measurement system.
2. Measurement procedure In this feasibility study, we first show the sensitivity of the method to detect silica NPs that are dispensed into the culture medium in different concentrations. For this purpose we used a simple two plate capacitor configuration (Fig. 1a) immersed directly into the beaker containing the suspension. For the experimental proof of NPs ingestion into cells we fabricated special coplanar electrodes (see Fig. 2) described below and fixed them on the bottom of each well of the 24-well plates. After that the wells were seeded with caco-2 cells maintained in a cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM). After 21 days of differentiation the cells are accrued on the electrodes (see Fig. 2c). Then the cells were treated with different Silica NPs concentrations from 25 μg/ml to 500 μg/ml for 24h. After that, the culture media was removed, and the cells were washed using Phosphate Buffered Saline (PBS) buffer to remove possible sedimented NPs from the cell surface. Next the coplanar electrodes were contacted with the spring probe (see Fig. 1b) and the electrical impedance was measured for various electrode combinations (see Fig. 3c).
II I a)
b)
c)
Fig. 2. (a) Shows the electrode design and the naming of the different electrodes; (b) Shows a detailed view of the electrodes containing the dimensioning; (c) Shows the Caco-2 cells grown on the electrodes. I) with Au and II) without Au
366
S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
3. Electrode design For the measurements a special electrode design was used to simplify the handling and offer the possibility to measure various positions in one sample without changing the electrode or the connections. The electrode itself is shown in Fig. 1 and consists of eight individually connectable electrodes which allow various electrode combinations for measurements at different sample areas. For the fabrication of the electrodes a 0.1 mm thick Polyethylenterephthalat (PET) film was used as a substrate. Circular shaped elements which directly fit into the 24well plate were cut out of the PET foil and a shadow mask was used to directly evaporate 30 nm thick gold (Au) structures as the measurement electrodes on them. The biocompatibility of the Au-electrodes was approved by the results from cytotoxicity biochemical assay (Lactate dehydrogenase (LDH) release). The segmented contact pads for the connections of the measurement electronics are placed on the outer boundary of the circular substrates and can be contacted with spring probes. In the middle of the electrode substrate is a transparent region to allow the use of a transmitted light microscope, which is very important for an optical control during the growth phase (see Fig. 2c). The electrodes are placed directly on the bottom of the 24-well plate. The electrical connection of the substrate is performed using eight spring probes arranged in a circle (see Fig. 1b) and mounted into a buckler. The buckler fits on the used 24-well plate and guaranties a good connection of the spring probes on the gold electrodes. 4. Readout electronics For the measurement of the impedance spectrum an Agilent 4294A Precision Impedance Analyzer was used. This measurement system works with an auto balancing bridge principle [4] and provides a four terminal connection interface. This interface has two connectors for the current path (HC and LC) and two connectors for the potential measurements (HP and LP). Between the HC and the LC connectors an alternating current with constant amplitude is applied by the measurement system. The other two ports (HP and LP) are used for the potential measurements. To keep the advantages of the four terminal measurement principle the connectors H C and HP should be connected together as close to the device under test (DUT) as possible, the same applies also for the LC and LP connectors. For the implementation of the automatic connection of arbitrary electrode combinations, two matrix switches from Maxim Integrated were used (MAX14661). This 16:2 analog matrix switch allows it to connect each of the 16 pins to any of the common pins simultaneously in any combination [5]. For the circuit all of the four terminal connections need to be switchable in any combination, therefore two of the matrix switches were combined to a 16:4 system. This connection method allows neglecting the “on” resistance RON and keeping the advantages of a four wire measurement over a large frequency range (40 Hz up to 10 MHz). For the measurement it is important to keep the current over the matrix switch below the maximum rating of 50 mA to guarantee correct measurement results. The control of the measurement system was implemented in MATLAB controlling the impedance analyzer via an Ethernet interface to allow an automatic sequence of impedance measurements for various electrode combinations. The communication with the matrix switches was implemented using a μController as USB to Serial Peripheral Interface (SPI) converter. 5. Measurements As a first measurement the direct influence of the nanoparticles in the medium was measured with a parallel-plate capacitor and compared to the results of the coplanar electrode setup. Fig 3 a) and b) shows the results for different concentrations of NPs with both setups. As mentioned above, the coplanar setup allows for a large variety of electrode connections. The electrodes a, b, c and d are “center electrodes”, i.e. they form a segmented ring in the center of the well (see Fig. 2a). The other electrodes e, f, g and h form are placed outside of these center electrodes as shown in Fig. 2a. Table 1 summarizes the used options in measuring impedances between two electrodes or groups of these electrodes. In case of “single electrode combinations” two single electrodes are selected and the impedance between them is measured where only the center electrodes are used. In case of “double electrode combinations” the impedance between a single electrode and two other connected neighboring electrodes is
367
S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
determined. In that manner, the measurement area in the well can be spatially shifted. In the measurements given in Fig 3b the average of single electrode measurements in group A (see Table 1) is shown which represents the situation at the center of the well. In Fig 3c, as an example for the spatial variation, all impedances are shown in one plot. The spectra for each group (A, B, C, D and E, see Table 1) are shown in the same color and they virtually coincide such that they appear as a single characteristic. Table 1. Measured electrode combinations. Combinations
Groups A)
Single electrode combinations B)
C)
gap between adjacent center electrodes gap between opposite center electrodes
Two center electrodes
Double electrode combination D)
Full electrode measurement
a)
E)
One center electrode
All electrodes
b)
Number in sequence
Terminals: HC, HP
Terminals: LC, LP
1
a
b
2
b
c
3
c
d
4
d
a
5
a
c
6
b
d
7
a, b
e
8
b, c
f
9
c, d
g
10
d, a
h
11
a
h, e
12
b
e, f
13
c
f, g
14
d
g, h
15
a, b, c, d
e, f, g, h
c)
Fig. 3. (a) Measurement results using the parallel-plate capacitor sensor setup shown in Fig. 2 a) for the nanoparticles dispensed into the culture medium with different concentrations at 3.2 kHz; (b) Measured impedance spectra for different nano particle concentrations using the coplanar electrode configuration group A (see Table 1); (c) Measurement for all different electrode configurations specified in Table 1 for one sample (particle concentration 50 μg/ml).
It is clearly visible that the parallel-plate capacitor provides more sensitive results to the particle concentration whereas the sensitive area of the coplanar design is concentrated in the gap between the electrodes thus yielding smaller signals. However, with the co-planar electrode arrangement not the whole volume is sensed but only a small layer above the electrodes which allows to detect a potentially non-uniform spatial distribution of the cells, which was not present in the measurement shown, though. This present uniformity in the particle and cell distribution is also indicated by the aforementioned coinciding spectra within each group (A, B, C, D, and E) of electrode combinations as shown in Fig 3c.
368
S. Clara et al. / Procedia Engineering 87 (2014) 364 – 368
A disadvantage of the measurements method is that, in the current design, the connections to the coplanar electrodes by means of the spring probes are realized in the sample, i.e. at the bottom of the well. Therefore the reproducibility cannot be guaranteed and the measurements for different equally filled wells tend to vary by random offsets due to variations in contact resistances. 6. Conclusion We presented measurement results for different doses of silica NPs supplemented in the cell culture media, measured with both the parallel-plate capacitor and the coplanar electrode design. Both type of electrodes show a clear dependency of the frequency dependent electrical impedance on the NPs concentration where the parallel-plate capacitor design provides more reproducible results due to the more homogeneous field distribution and the averaging over a relatively large volume. The measurements with the coplanar electrodes offer the possibility of spatially resolved measurements in small sample wells and hence to probe spatially defined areas of the cell culture, but suffer from the fact that the electrical connections between spring probes and electrodes can be affected by sample residues in the contact area. 7. Outlook The previously mentioned problems with the connection of the electrodes and the hardly controllable deviations in contact resistance and in cell population between the substrates in different wells require the development of an improved measurement principle where each of the cell culture wells is monitored separately over the time. This measurement method will allow using the relative changes of the spectra to monitor the cell behavior in response to NPs. Acknowledgements This work was supported by the “REGIO 13 - Impulse für OÖ” program, the “Austrian Center of Competence in Mechatronics” (ACCM) funded by the Austrian Research Promotion Agency (FFG) and by State of Upper Austria. References [1] [2] [3]
[4] [5]
EFSA - European Food Safety Authority. 2009. The potential risks arising from nanoscience and nanotechnologies on food and feed safety (EFSA-Q-2007-124a). The EFSA Journal 958, 1-39 Moghimi SM, Hunter AC, Murray JC: Nanomedicine: current status and future prospects. FASEB J 2005, 19:311-330. Environ Sci Technol. 2005 Dec 1;39(23):9370-6. Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Limbach LK1, Li Y, Grass RN, Brunner TJ, Hintermann MA, Muller M, Gunther D, Stark WJ Agilent Impedance Measurement Handbook A guide to measurement technology and techniques 4th Edition, 2009-2013 Datasheet: MAX14661 Beyond-the-Rails 16:2 Multiplexer, maxim integrated